Compositions and methods of enhancing immune responses to enteric pathogens

ABSTRACT

Vaccine vectors capable of eliciting an immune response to enteric bacteria and methods of using the same are provided. The vaccine vectors include a polynucleotide encoding a PAL polypeptide. The PAL polypeptide may be expressed on the surface of the vaccine vector. The vaccine vector may also include a second polypeptide encoding an immunostimulatory polypeptide such as a CD154 polypeptide or an HMGB1 polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. application Ser. No. 16/531,893, filed Aug. 5, 2019, and issued as U.S. Pat. No. 10,716,840 on Jul. 21, 2020 which is a continuation of U.S. 371 patent application Ser. No. 14/776,986, filed Sep. 15, 2015, and issued as U.S. Pat. No. 10,376,571 on Aug. 13, 2019, which is a national stage filing under 35 U.S.C. 371 of International Application No. PCT/US2014/027416, filed Mar. 14, 2014, which claims the benefit of priority of U.S. Provisional Patent Application No. 61/790,301, filed Mar. 15, 2013, all of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

This application is being filed electronically and includes an electronically submitted Sequence Listing in .txt format. The .txt file contains a sequence listing entitled “2014-03-14_5658-00203_ST25.txt” created on Mar. 14, 2014 and is 31,093 bytes in size. The Sequence Listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.

INTRODUCTION

Bacterial infections still pose a significant health hazard to humans and agricultural and domesticated animals. The increase in antibiotic resistance has increased the need to move away from use of antibiotics in agriculture and the need to develop alternative methods of controlling bacterial infections and bacterial contamination of the human food supply. Salmonella and E. coli are commonly reported bacterial causes of human food-borne infections worldwide, and epidemiological evidence indicates that meat products including poultry and poultry products are a significant source of human infection. In the United States, an estimated 1.4 million cases of human Salmonellosis are reported annually. Of these cases, S. enterica serovars Enteritidis (SE) and Typhimurium (ST) are the most commonly isolated, although a number of other serovars have also been shown to cause enteritis in humans. Other gram negative bacteria responsible for significant infection rates include Shigella spp, Vibrio spp, Erwinia spp, Klebsiella spp, Citrobacter spp, Yersinia spp, Providencia spp and similar bacteria. Novel means to control these bacterial infections are needed.

SUMMARY

A vaccine vector comprising a first polynucleotide sequence encoding a PAL polypeptide is disclosed. The PAL polypeptide is a heterologous, non-natively expressed, recombinant polypeptide in the vaccine vector. The PAL polypeptide is selected from SEQ ID NO: 1, a sequence with 90% identity to SEQ ID NO: 1, such as SEQ ID NO: 6, or an immunogenic fragment thereof at least six amino acids long. The polypeptide may be expressed on the surface of the vaccine vector. The immunogenic fragment of SEQ ID NO: 1 may comprise SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 36 or SEQ ID NO: 37. The vaccine vector may also comprise a second polypeptide sequence encoding an immunostimulatory polypeptide. The immunostimulatory polypeptide may also be expressed on the surface of the vaccine vector. The immunostimulatory polypeptide may be a CD154 polypeptide capable of binding CD40 or an HMGB1 polypeptide. The CD154 polypeptides include fewer than 50 amino acids and comprise amino acids 140-149, or a homolog thereof.

Vaccines according to the present invention may be comprised within a vector, such as a virus, yeast, bacterium, or liposome. In one aspect, the vaccines include polynucleotides encoding polypeptides of SEQ ID NO: 42, 44 or 46 or a sequence having 90% identity to one of these sequences. Pharmaceutical compositions may be comprised of the vaccine vectors described herein and a pharmaceutically acceptable carrier.

In still another aspect, methods of enhancing the immune response against a gram-negative bacterium in a subject by administering a vaccine vector described herein to the subject are provided. The enhanced immune response may be an enhanced antibody response, an enhanced T cell response or a combination thereof.

In a still further aspect, methods of reducing morbidity or mortality associated with infection with a gram-negative bacterium in a subject by administering a vaccine vector as described herein to the subject are provided. The vaccine vector is capable of reducing the morbidity and mortality associated with subsequent infection with a gram-negative bacterium in subjects administered the vaccine vector as compared to controls.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the PAL sequence-specific serum IgG antibody levels as determined by ELISA with synthetic PAL-BSA as the coating antigen on day 17 post vaccination by oral gavage with either saline, or Bacillus backbone (BS BB) or PAL-vectored BS vaccine (BS PAL) candidates at 10⁸ cfu/chick. The results are presented as mean S/P ratios±SEM (n=10). Groups with different upper case letters are significantly different using an ANOVA (P<0.05).

FIG. 2 is a graph showing the PAL sequence-specific ileal sIgA antibody levels as determined by ELISA with synthetic PAL-BSA as the coating antigen on day 17-post vaccination by oral gavage with either saline, or Bacillus backbone (BS BB) or PAL-vectored BS vaccine candidates (BSNNP) at 10⁸ cfu/chick. The results are presented as mean S/P ratios±SEM (n=10). Groups with different upper case letters are significantly different using an ANOVA (P<0.05).

FIG. 3 is a graph in which Salmonella typhimurium was enumerated in chicks receiving saline, BSBB or PAL-BS construct vectored vaccine (BSNNP) at 10⁸ cfu/chick using conventional microbiological plate counting at 17 and 21 days post hatch. All groups received ST challenge dose of 1×10⁸ cfu/ml on day 11 post-hatch. The results are presented as mean log₁₀ cfu/gram of cecal content+SEM (n=10). Groups with different upper case letters are significantly different by ANOVA (P<0.05).

FIG. 4 is a graph showing the affinity of two monoclonal antibodies (2B5 and 1B2) as compared to control (PBST) for the indicated hexapeptides of PAL.

FIG. 5 is a graph showing the Salmonella Heidelberg colony forming units (cfu) per gram isolated from the ceca of 21-day-old broilers after vaccination with the indicated vaccine strain or control. Groups with different upper case letters are significantly different by ANOVA (P<0.05).

FIG. 6 is a graph showing the Salmonella Heidelberg colony forming units (cfu) per gram isolated from the ceca of 21-day-old and from 33-day-old broilers after vaccination with the indicated vaccine strain or control. From left to right the graph shows control vaccinated chickens at 21 and 33 days after challenge, or chickens vaccinated with Salmonella enteriditis with the inserts arranged from N to C terminal as CJ0113-PAL-HMGB1; CJ0113-mHMGB1-mPAL or mHMGB1-CJ0113-PAL, wherein m is an indication of a mutation in the sequence of the protein. Groups with an asterisk are significantly different by ANOVA (P<0.05).

FIG. 7 is a graph showing the Salmonella Heidelberg positive percent recovery from the ceca of 28 day old broilers after vaccination with the indicated vaccine strain or controls. Groups with different upper case letters are significantly different by ANOVA (P<0.05).

FIG. 8 is a graph showing the percent phagocytosis of the indicated vaccine strains by murine macrophages. Groups with different upper case letters are significantly different by ANOVA (P<0.05).

DETAILED DESCRIPTION

Conventional vaccines against gram-negative bacteria are generally based on live/attenuated bacteria that are delivered in controlled numbers often via injection. Gram-negative bacteria are quite diverse and antigenic diversity among the different species of bacteria and even among different strains within the same species has made vaccination against more than a single strain or serovar difficult. Recombinant vaccines have been developed but because of the antigenic diversity are generally restricted to enhancing an immune response to a single species or even a single strain of bacteria. A vaccine capable of protecting against multiple serovars and indeed against more than one species of gram-negative bacteria would be optimal. In addition, a vaccine that could be given orally would make administration cheaper and compliance more likely. A vaccine comprising a highly conserved region of PAL, a peptidoglycan-associated lipoprotein found broadly on gram-negative organisms, is provided.

Recombinant DNA technologies enable relatively easy manipulation of many yeast, bacterial and viral species. Some microorganisms are mildly pathogenic or non-pathogenic, but are capable of generating a robust immune response. These microorganisms make attractive vaccine vectors for eliciting an immune response to antigens recombinantly expressed in the vector. Vaccines vectored by microorganisms may mimic a natural infection, help produce robust and long lasting mucosal immunity, and may be relatively inexpensive to produce and administer. Many of these vaccine vectors can be administered orally which reduces the cost and need for professionals for administration and lowers resistance to administration. In addition, such vectors can often carry more than one antigen and have potential to provide protection against multiple infectious agents.

A vaccine includes any composition comprising a polynucleotide encoding an antigenic polypeptide that is capable of eliciting an immune response to the polypeptide. A vaccine vector is a composition that can be engineered to carry antigens and optionally other immunostimulatory polypeptides and may also comprise an adjuvant or be administered with an adjuvant to further increase the immune response to the parasite and provide better protection from morbidity and mortality associated with a subsequent infection. The use of vectors, such as bacterial, viral or yeast vectors, for vaccination and generation of immune responses against enteric pathogens is disclosed herein. The enteric pathogens may include, but are not limited to E. coli, Salmonella and the other enteric microorganisms disclosed in Table 1 in the Examples. The immune responses after administration of the vaccine vectors described herein need not be fully protective, but may decrease the morbidity or percentage mortality (i.e. likelihood of mortality) associated with subsequent infection with an enteric pathogen.

In one aspect, a vaccine vector comprising a first polynucleotide sequence encoding at least one of SEQ ID NO: 1-6, 32, 36 or 37 or an immunogenic fragment at least six amino acids long of any one of these sequences is provided. The vaccine vector may also include a second polynucleotide encoding an immunostimulatory polypeptide is provided. Suitably the PAL polypeptide or immunogenic fragments thereof and the immunostimulatory polypeptide are expressed on the surface of the vaccine vector. The immunogenic fragments of the polypeptide of SEQ ID NO: 1 may comprise any one of or a combination of SEQ ID NOs: 2-5 or 36-40 or any other fragment of at least six amino acids. For example, the antigenic polypeptide may comprise, may consist essentially of or may consist of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 37 or an immunogenic fragment or combination of any of these SEQ ID NOs.

An immunogenic fragment of the antigenic polypeptide may be a sequence that is at least 6, 8, 10, 12, 14, 16, 18 or 20 amino acids long and has at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% percent identity to the SEQ ID NOs provided herein. A vaccine includes any composition comprising a polynucleotide encoding an antigenic polypeptide that is capable of eliciting an immune response to the polypeptide in a subject administered the vaccine. The use of vectors, such as bacterial vectors, for vaccination and generation of immune responses against enteric bacteria, including but not limited to Salmonella spp, Escherichia spp, Shigella spp, Vibrio spp, Erwinia spp, Klebsiella spp, Citrobacter spp, Yersinia spp, Providencia spp or similar bacteria such as those listed in Table 1 is disclosed.

Polynucleotides encoding the antigenic polypeptides provided herein and other antigens from any number of pathogenic organisms may be inserted into the vector and expressed in the vector. The expression of these polynucleotides by the vector will allow generation of an immune response to the antigenic polypeptides following immunization of the subject. The polynucleotides may be inserted into the chromosome of the vector or encoded on plasmids or other extrachromosomal DNA. Those of skill in the art will appreciate that numerous methodologies exist for obtaining expression of polynucleotides in vectors such as Salmonella or Bacillus. The polynucleotides may be operably connected to a promoter (e.g., a constitutive promoter, an inducible promoter, etc.) by methods known to those of skill in the art. Suitably, polynucleotides encoding antigenic polypeptides are inserted into a vector, e.g., a bacterial vector, such that the polynucleotide is expressed.

The polynucleotides encoding PAL or other antigenic polypeptides may be inserted in frame in a polynucleotide encoding a transmembrane protein. The polynucleotide encoding the antigenic polypeptide may be inserted into the vector polynucleotide sequence to allow expression of the antigenic polypeptide on the surface of the vector. For example, the polynucleotide encoding antigenic polypeptide may be inserted in frame into the vector polynucleotide in a region encoding an external loop region of a transmembrane protein such that the vector polynucleotide sequence remains in frame. In one embodiment, the first polynucleotide encoding the antigenic polypeptide may be inserted into loop 9 of the lamB gene of Salmonella as described in the Examples. Alternatively, the polynucleotide could be inserted in a polynucleotide such as the cotB gene of Bacillus.

In another embodiment, the first polynucleotide is inserted into or at a surface exposed end of a protein that is attached to the cell wall, but is not a transmembrane protein. The protein may be a secreted protein that is anchored or attached to the cell wall via a protein or lipid anchor. For examples, the polynucleotide may be inserted at the 3′ end of the fibronectin binding protein (FbpB) of Bacillus subtilis. Alternatively, the first polynucleotide encoding the antigenic polypeptide may be inserted into a polynucleotide encoding a secreted polypeptide.

Those of skill in the art will appreciate that the polynucleotide encoding the antigenic polypeptide could be inserted in a wide variety of vector polynucleotides to provide expression and presentation of the antigenic polypeptide to the immune cells of a subject treated with the vaccine. The polynucleotide encoding the antigenic polypeptide may be included in a single copy or more than one copy. The multiple copies may be inserted in a single location or more than one location within the vaccine vector chromosome or extrachromosomally.

Suitably the first polynucleotide encodes SEQ ID NO: 1, SEQ ID NO: 6 or an immunogenic fragment thereof at least six or more amino acids such as SEQ ID NO: 2-5, or 36-40. The vector may include more than one copy of the first polynucleotide or may include multiple antigenic polynucleotides targeted to the same or different pathogens. In the Examples, SEQ ID NOs: 1-6, 32, 36 and 37 were shown to be immunogenic. SEQ ID NOs: 1 (EGHADERGTPEYNISLGER) and 8 (TVEGHADERGTPEYNISLG) are incorporated into a Bacillus or Salmonella vector in the Examples. The combination of epitopes from more than one polypeptide from a single pathogen or target or the combination of epitopes from distinct pathogens or targets is specifically contemplated. The polynucleotides may be inserted into the vector separately or may be inserted as a fusion protein containing more than a single epitope. In the Examples, SEQ ID NOs: 1 (PAL) and 31 (CJ0113) were incorporated into a Bacillus vector (see SEQ ID NO: 42, 44 and 46 and the Examples). Suitably, the portion of the antigenic polypeptide inserted into the vector is immunogenic. An immunogenic fragment is a peptide or polypeptide capable of eliciting a cellular or humoral immune response or capable of reducing morbidity or mortality associated with subsequent infection with the target pathogen or a related pathogen.

An antigenic polypeptide includes any polypeptide that is immunogenic. The antigenic polypeptides include, but are not limited to, antigens that are pathogen-related, allergen-related, tumor-related or disease-related. Pathogens include viral, parasitic, fungal and bacterial pathogens as well as protein pathogens such as the prions. The antigenic polypeptides may be full-length proteins or portions thereof. It is well established that immune system recognition of many proteins is based on a relatively small number of amino acids, often referred to as the epitope. Epitopes may be only 4-8 amino acids. Thus, the antigenic polypeptides described herein may be full-length sequences, four amino acid long epitopes or any portion between these extremes. In fact the antigenic polypeptide may include more than one epitope from a single pathogen or protein. The antigenic polypeptides may have at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% percent identity to the SEQ ID NOs provided herein. Suitably, an antigenic fragment of a polypeptide may be four, five, six, seven, eight, nine, ten, twelve, fifteen, seventeen or more consecutive amino acids, of SEQ ID NO: 1-6, 32, 36 or 37.

Multiple copies of the same epitope or multiple epitopes from different proteins may be included in the vaccine vector. The epitopes in the vaccine vector may be related and homologous to allow targeting of multiple related pathogens with a single vaccine vector. It is envisioned that several epitopes or antigens from the same or different pathogens or diseases may be administered in combination in a single vaccine vector to generate an enhanced immune response against multiple antigens. Recombinant vaccine vectors may encode antigens from multiple pathogenic microorganisms, viruses or tumor associated antigens. Administration of vaccine vectors capable of expressing multiple antigens has the advantage of inducing immunity against two or more diseases at the same time, providing broader protection against multiple strains of a single pathogen or a more robust immune response against a single pathogen. In the Examples, the vaccine vectors included the PAL antigenic polypeptide of SEQ ID NO: 1 and a Campylobacter antigenic polypeptide of SEQ ID NO: 31 already demonstrated to be effective to enhance the immune response to Campylobacter in International Patent Publication No. WO2011/156619.

Those of skill in the art will appreciate that the antigenic polypeptides from other pathogens may be used in the vaccine vectors to enhance the immune response against more than one pathogen by a single vaccine. It would be advantageous to administer a single vaccine directed against multiple pathogens. A vaccine capable of eliciting an immune response to an enteric pathogen, such as E. coli, in combination with Influenza, Salmonella, Campylobacter or other pathogens is envisioned. For example, the second antigenic polypeptide may be an Influenza polypeptide, suitably it is an Influenza H5N1 polypeptide or a polypeptide associated with multiple strains of the Influenza virus such as a polypeptide of the Influenza M2 protein. The ectodomain of the Influenza A virus M2 protein, known as M2e, protrudes from the surface of the virus. The M2e portion of the M2 protein contains about 24 amino acids. The M2e polypeptide varies little from one isolate to the next within Influenza. In fact, only a few naturally occurring mutations in M2e have been isolated from infected humans since the 1918 flu epidemic. In addition, influenza viruses isolated from avian and swine hosts have different, yet still conserved, M2e sequences. For reviews of the M2e polypeptide sequences isolated from human, avian and swine hosts see Liu et al., Microbes and Infection 7:171-177 (2005) and Reid et al., J. Virol. 76:10717-10723 (2002) each of which are incorporated herein by reference in its entirety. Suitably the entire M2e polypeptide may be inserted into the vaccine vector or only a portion may be used. An eight amino acid polypeptide (LM2 having amino acid sequence: EVETPIRN, SEQ ID NO: 9 or its variant M2eA having amino acid sequence EVETPTRN, SEQ ID NO: 10) was incorporated into the vaccine vector and demonstrated to produce an antibody response after administration to chickens. See U.S. Publication No. 2011/0027309 which is incorporated herein by reference in its entirety.

Other suitable epitopes for inclusion in a vaccine vector to enhance an immune response to Influenza A include, but are not limited to, polypeptides of the hemagglutinin (HA) or the nuclear protein (NP) of Influenza A. For example, the peptides of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14 may be included in a vaccine vector. One of skill in the art will appreciate that any of these sequences may be used in combination with any other epitope including epitopes derived from other pathogens or antigens.

For example, the PAL antigenic polypeptide provided herein may be combined with other antigenic polypeptides from gram negative bacteria such as those provided in U.S. Patent Publication No. US2011/0159026 or International Publication No. WO 2011/156619, both of which are incorporated by reference herein in their entireties. The combination of multiple antigenic polypeptides, one of which provides broad immunity to multiple gram negative bacteria and others that are more specific to particular gram negative bacteria may provide superior protection from subsequent infection.

Immunostimulatory molecules included as part of the vaccine vector could potentially activate parts of the immune system critical to long-lasting protection. Immunostimulatory polypeptides may be polypeptides capable of stimulating a naïve or adaptive immune response. The immunostimulatory polypeptides are not natively associated with the vaccine vector and are polypeptides natively associated with a vertebrate immune system, such as that of the subject to which the vaccine will be administered. Two immunostimulatory polypeptides are described herein, namely CD154 and High Mobility Group Box 1 (HMGB1) polypeptides, but one of skill in the art will appreciate that other immunostimulatory polypeptides could be used or alternatively could be used in combination with those described herein.

Additional polynucleotides encoding polypeptides involved in triggering the immune system may also be included in a vaccine vector. The polynucleotides may encode immune system molecules known for their stimulatory effects, such as an interleukin, Tumor Necrosis Factor, interferon, or another polynucleotide involved in immune-regulation. The vaccine may also include polynucleotides encoding peptides known to stimulate an immune response, such as the CD154 or HMGB1 polypeptides described herein.

HMGB1 is secreted by activated macrophages and damaged cells, and acts as a cytokine mediator of inflammation, affecting the innate immune response. Portions of the HMGB1 sequence have been included in the vaccine vectors described in the Examples. The HMGB1 (High Mobility Group Box-1) protein was first identified as a DNA-binding protein critical for DNA structure and stability. It is a ubiquitously expressed nuclear protein that binds DNA with no sequence specificity. The protein is highly conserved and found in plants to mammals. The zebrafish, chicken and human HMGB1 amino acid sequences are provided in SEQ ID NO: 23, SEQ ID NO: 15 and SEQ ID NO: 22, respectively. The sequence throughout mammals is highly conserved with 98% amino acid identity and the amino acid changes are conservative. Thus an HMGB1 protein from one species can likely substitute for that from another species functionally. The full-length HMGB1 protein or a portion thereof may be used as the HMGB1 polypeptide in the vaccine vectors described herein. HMGB1 has two DNA binding regions termed A box as shown in SEQ ID NO: 16 and 17 and B box as shown in SEQ ID NO: 18 and 19. See Andersson and Tracey, Annu. Rev. Immunol. 2011, 29:139-162, which is incorporated herein by reference in its entirety.

HMGB1 is a mediator of inflammation and serves as a signal of nuclear damage, such as from necrotic cells. HMGB1 can also be actively secreted by cells of the monocyte/macrophage lineage in a process requiring acetylation of the protein, translocation across the nucleus and secretion. Extracellular HMGB1 acts as a potent mediator of inflammation by signaling via the Receptor for Advanced Glycated End-products (RAGE) and via members of the Toll-like Receptor family (TLR), in particular TLR4. The RAGE binding activity has been identified and requires the polypeptide of SEQ ID NO: 20. TLR4 binding requires the cysteine at position 106 of SEQ ID NO: 15, which is found in the B box region of HMGB1.

The inflammatory activities of HMGB1 do not require the full-length protein and functional fragments have been identified. The B box has been shown to be sufficient to mediate the pro-inflammatory effects of HMGB1 and thus SEQ ID NO: 18 and 19 are HMGB1 polypeptides or functional fragments thereof within the context of the present invention. In addition, the RAGE binding site and the pro-inflammatory cytokine activity have been mapped to SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Thus, these polypeptides are functional fragments of HMGB1 polypeptides in the context of the present invention.

Those of skill in the art are capable of identifying HMGB1 polypeptides and fragments thereof capable of stimulating pro-inflammatory cytokine activity, using methods such as those in International Publication No. WO02 092004, which is incorporated herein by reference in its entirety. Suitably, the HMGB1 polypeptide includes the RAGE binding domain at amino acids 150-183 of SEQ ID NO:15 (SEQ ID NO: 20 or a homolog thereof) and the pro-inflammatory cytokine activity domain between amino acids 89-109 of SEQ ID NO: 15 (SEQ ID NO: 21 or a homolog thereof). In particular, HMGB1 polypeptides and functional fragments or homologs thereof include polypeptides identical to, or at least 99% identical, at least 98% identical, at least 95% identical, at least 90% identical, at least 85% identical, or at least 80% identical to the HMGB1 polypeptides of SEQ ID NOs: 15 or 16-23.

As described in more detail below, a vaccine vector may include a CD154 polypeptide that is capable of binding CD40 in the subject and stimulating the subject to respond to the vector and its associated antigen. Involvement of dendritic cells (DCs) is essential for the initiation of a powerful immune response as they possess the unique ability to activate naïve T cells, causing T cell expansion and differentiation into effector cells. It is the role of the DC, which is an antigen presenting cell (APC) found in virtually all tissues of the body, to capture antigens, transport them to associated lymphoid tissue, and then present them to naïve T cells. Upon activation by DCs, T cells expand, differentiate into effector cells, leave the secondary immune organs, and enter peripheral tissues. Activated cytotoxic T cells (CTLs) are able to destroy virus-infected cells, tumor cells or even APCs infected with intracellular parasites (e.g., Salmonella) and have been shown to be critical in the protection against viral infection. CD40 is a member of the TNF-receptor family of molecules and is expressed on a variety of cell types, including professional antigen-presenting cells (APCs), such as DCs and B cells. Interaction of CD40 with its ligand CD154 is extremely important and stimulatory for both humoral and cellular immunity. Stimulation of DCs via CD40, expressed on the surface of DCs, can be simulated by anti-CD40 antibodies. In the body, however, this occurs by interaction with the natural ligand for CD40 (i.e. CD154) expressed on the surface of activated T-cells. Interestingly, the CD40-binding regions of CD154 have been identified. The CD40-binding region of CD154 may be expressed on the surface of a vector, such as a Salmonella or Bacillus vector, and results in an enhanced immune response against a co-presented peptide sequence as shown in the Examples provided herein and in U.S. Patent Publication No. 2011/0027309, which is incorporated herein by reference in its entirety. A CD154 polypeptide may be a portion of CD154 full-length protein or the entire CD154 protein. Suitably, the CD154 polypeptide is capable of binding CD40.

As discussed above, a CD154 polynucleotide encoding a CD154 polypeptide that is capable of enhancing the immune response to the antigen may be included in the vaccine. Suitably, the CD154 polypeptide is fewer than 50 amino acids long, more suitably fewer than 40, fewer than 30 or fewer than 20 amino acids in length. The polypeptide may be between 10 and 15 amino acids, between 10 and 20 amino acids or between 10 and 25 amino acids in length. The CD154 sequence and CD40 binding region are not highly conserved among the various species. The CD154 sequences of chicken and human are provided in SEQ ID NO: 24 and SEQ ID NO: 25, respectively.

The CD40 binding regions of CD154 have been determined for a number of species, including human, chicken, duck, mouse and cattle and are shown in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively. Although there is variability in the sequences in the CD40 binding region between species, the human CD154 polypeptide was able to enhance the immune response in chickens. Therefore, one may practice the invention using species specific CD154 polypeptides or a heterologous CD154 polypeptide. Thus the CD154 polypeptides of SEQ ID NO: 24-30 may be included in a vaccine vector or a polypeptide at least 99, 98, 97, 96, 95, 93, 90 or 85% identical to the sequences of SEQ ID NO: 24-30 may be included in a vaccine vector.

The polypeptide from CD154 stimulates an immune response at least in part by binding to its receptor, CD40. A polypeptide homologous to the CD154 polypeptide which is expressed on immune cells of the subject and which is capable of binding to the CD40 receptor on macrophages and other antigen presenting cells. Binding of this ligand-receptor complex stimulates macrophage (and macrophage lineage cells such as dendritic cells) to enhance phagocytosis and antigen presentation while increasing cytokine secretions known to activate other local immune cells (such as B-lymphocytes). As such, molecules associated with the CD154 peptide are preferentially targeted for immune response and expanded antibody production.

The antigenic polypeptides and the immunostimulatory polypeptides are delivered via a vaccine vector. The vaccine vectors may be bacterial, yeast, viral or liposome-based vectors. Potential vaccine vectors include, but are not limited to, Bacillus (Bacillus subtilis), Salmonella (Salmonella enteritidis), Shigella, Escherichia (E. coli), Yersinia, Bordetella, Lactococcus, Lactobacillus, Streptococcus, Vibrio (Vibrio cholerae), Listeria, yeast such as Saccharomyces, or Pichia, adenovirus, poxvirus, herpesvirus, alphavirus, and adeno-associated virus. Live bacterial, yeast or viral vaccine vectors may still pose risks to immunocompromised individuals and require additional regulatory scrutiny. Thus use of vectors that are killed or inactivated or qualify as Generally Recognized As Safe (GRAS) organisms by the Food and Drug Administration (FDA) is desirable. The problem is generating a robust immune response using such vectors. Methods of inactivating or killing bacterial, yeast or viral vaccine vectors are known to those of skill in the art and include, but are not limited to methods such as formalin inactivation, antibiotic-based inactivation, heat treatment and ethanol treatment. By including an immunostimulatory polypeptide such as HMGB1 (high mobility group box 1) polypeptide on the surface of the vaccine vector we can generate a robust immune response against an antigenic polypeptide using a Bacillus spp. vector or other GRAS vector. In fact, such vectors can be inactivated such that it cannot replicate and still elicit a robust immune response after administration. The vaccine vectors may be wild-type bacteria, yeasts or viruses that are not pathogenic. Alternatively the vectors may be attenuated such that the vector has limited ability to replicate in the host or is not capable of growing without supplemented media for more than a few generations. Those of skill in the art will appreciate that there are a variety of ways to attenuate vectors and means of doing so.

At least a portion of the antigenic polypeptide and at least a portion of the immunostimulatory polypeptide are present or expressed on the surface of the vaccine vector. Present on the surface of the vaccine vector includes polypeptides that are comprised within an external loop of a transmembrane protein, interacting with, e.g., covalently or chemically cross-linked to, a transmembrane protein, a membrane lipid or membrane anchored carbohydrate or polypeptide. A polypeptide can be comprised within a transmembrane protein by having the amino acids comprising the polypeptide linked via a peptide bond to the N-terminus, C-terminus or anywhere within the transmembrane protein (i.e. inserted between two amino acids of the transmembrane protein or in place of one or more amino acids of the transmembrane protein (i.e. deletion-insertion)). Suitably, the polypeptides may be inserted into an external loop of a transmembrane protein. Suitable transmembrane proteins are srtA, cotB and lamB, but those of skill in the art will appreciate many suitable transmembrane proteins are available. Polypeptides may be linked to a membrane or cell wall anchored protein or lipid such that the antigenic polypeptide and the immunostimulatory polypeptide are expressed on the surface of the vaccine vector.

As described above, polynucleotides encoding the antigenic or immunostimulatory polypeptides may be inserted into the chromosome of the vector or maintained extrachromosomally (e.g., on a plasmid, BAC or YAC). One of skill in the art will appreciate that these polynucleotides can be inserted in frame in a variety of polynucleotides and expressed in different parts of the vector or may be secreted. The polynucleotide encoding the immunostimulatory polypeptide capable of enhancing the immune response to the antigenic polypeptide may also encode the antigenic polypeptide. The polynucleotide encoding the antigenic polypeptide may be linked to the polynucleotide encoding the immunostimulatory polypeptide, such that in the vector, the two polypeptides are portions of the same polypeptide. In the Examples, a polynucleotide encoding the antigenic polypeptide also encodes the immunostimulatory polypeptide. In one embodiment, the two polynucleotides encoding the polypeptides are both inserted in frame in loop 9 of the lamB gene of Salmonella enteritidis or another vaccine vector. Those of skill in the art will appreciate that bacterial polynucleotides encoding other transmembrane proteins and other loops of the lamB gene may also be used.

Alternatively, the polynucleotide encoding the antigenic polypeptide and/or the immunostimulatory polypeptide may be inserted into a secreted polypeptide that is displayed or presented on the surface of the vaccine vector through association with a protein, lipid or carbohydrate on the surface of the vaccine vector. Those of skill in the art will appreciate that the polynucleotide encoding the antigenic polypeptide and/or the immunostimulatory polypeptide could be inserted in a wide variety of vaccine vector polynucleotides to provide expression and presentation of the antigenic polypeptide and/or the immunostimulatory polypeptide to the immune cells of a subject treated with the vaccine vector. The coding region of the PAL antigenic polypeptide and the immunostimulatory polypeptide can be fused to the C-terminus of the Staphylococcus aureus fibronectin binding protein containing a sorting motif for sortase from Listeria. This allows the secreted proteins to be anchored on the cell wall of gram positive bacteria such as Bacillus. See Nguyen and Schumann, J Biotechnol (2006) 122: 473-482, which is incorporated herein by reference in its entirety. Other similar methods may also be used.

Alternatively, the polypeptides may be covalently or chemically linked to proteins, lipids or carbohydrates in the membrane, cell wall, or capsid if a viral vector is being used through methods available to persons of skill in the art. For example, di-sulfide bonds or biotin—avidin cross-linking could be used to present the antigenic and immunostimulatory polypeptides on the surface of a vaccine vector. Suitably, the antigenic polypeptide and the immunostimulatory polypeptide are part of a fusion protein. The two polypeptides may be directly linked via a peptide bond or may be separated by a linker, spacer, or a section of a third protein into which they are inserted. In the Examples, an amino acid spacer was used between the polypeptides. A spacer may be between 2 and 20 amino acids, suitably between 3 and 10 amino acids, suitably between 6 and 8 amino acids. Suitably the amino acids in the spacer have a small side chain and are not charged, such as glycine, alanine or serine. Spacers may have combinations of amino acid residues.

In the Examples, the vaccine vectors have the antigenic polypeptides (SEQ ID NO: 1 and SEQ ID NO: 31 (Campy CJ0113)) and the immunostimulatory polypeptide (HMGB1) encoded on the same polynucleotide and in frame with each other. See SEQ ID NO: 42, 44, and 46. Notably, in the Examples using a three amino acid spacer between each of the polypeptide fragments, the vaccine vector in which HMGB1 polypeptide was positioned on either the N- or C-terminal end of the vaccine vector insert resulted in the best protection against subsequent infection. The best performing vaccine vector had CJ0113 followed by PAL followed by HMGB1 (from N- to C-terminal or SEQ ID NO: 42). Thus the order or display of the antigens and immunostimulatory polypeptides on the surface of the vaccine vector may affect the immune response. In alternative embodiments, the immunostimulatory polypeptide and the antigenic polypeptide may be encoded by distinct polynucleotides. Those of skill in the art will appreciate that a variety of methods may be used to obtain expression of the antigenic polypeptide and the HMGB1 polypeptide on the surface of the vaccine vector. Such methods are known to those skilled in the art.

Compositions comprising the vaccine vector and a pharmaceutically acceptable carrier are also provided. A pharmaceutically acceptable carrier is any carrier suitable for in vivo administration. Suitably, the pharmaceutically acceptable carrier is acceptable for oral, nasal or mucosal delivery. The pharmaceutically acceptable carrier may include water, buffered solutions, glucose solutions or bacterial culture fluids. Additional components of the compositions may suitably include excipients such as stabilizers, preservatives, diluents, emulsifiers and lubricants. Examples of pharmaceutically acceptable carriers or diluents include stabilizers such as carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein-containing agents such as bovine serum or skimmed milk and buffers (e.g., phosphate buffer). Especially when such stabilizers are added to the compositions, the composition is suitable for freeze-drying or spray-drying. The vaccine vector in the compositions may not be capable of replication, suitably the vaccine vector is inactivated or killed prior to addition to the composition.

Methods of enhancing immune responses in a subject by administering a vaccine vector are also provided. The vaccine vector may contain a first polynucleotide encoding an antigenic PAL polypeptide of SEQ ID NO: 1-6, 32, 36, 37 or an immunogenic fragment thereof. The vaccine vector may also include a second polynucleotide encoding an immunostimulatory polypeptide. The immunostimulatory polypeptide is suitably a polypeptide natively associated with a vertebrate immune system and involved in stimulating an immune response. The immunostimulatory polypeptide may stimulate the native or adaptive immune response of the subject. Suitably a HMGB1 polypeptide or a CD154 polypeptide as described more fully above may be used as the immunostimulatory polypeptide. In the methods provided herein, the vaccine vector comprising an antigenic PAL polypeptide and optionally an immunostimulatory polypeptide is administered to a subject in an amount effective to enhance the/effect an immune response of the subject to the vaccine vector and in particular to the antigenic polypeptide and suitably to gram-negative bacteria such as Salmonella and E. coli.

The enhanced immune response may include an antibody or T cell response. Suitably the immune response is a protective immune response, but the immune response may not be fully protective, but may be capable of reducing the morbidity or mortality associated with infection. The immunostimulatory polypeptides may be used to enhance the immune response in the subject to any foreign antigen or antigenic polypeptide present in the vaccine vector in addition to the antigenic PAL polypeptide. One of skill in the art will appreciate that the immunostimulatory polypeptide could be used to enhance the immune response to more than one antigenic polypeptide present in a vaccine vector. Enhancing an immune response includes, but is not limited to, inducing a therapeutic or prophylactic effect that is mediated by the immune system of the subject. Specifically, enhancing an immune response may include, but is not limited to, enhanced production of antibodies, enhanced class switching of antibody heavy chains, maturation of antigen presenting cells, stimulation of helper T cells, stimulation of cytolytic T cells or induction of T and B cell memory.

Suitably, the vaccine vector contains a polynucleotide encoding a polypeptide including amino acids 150-183 and 89-109 of the HMGB1 polypeptide (SEQ ID NO: 15) or a homolog thereof. In the Examples, a 190 amino acid polypeptide of HMGB1 was used. Suitably, the polynucleotide encodes a HMGB1 polypeptide from the same species as the subject. Heterologous combinations of HMGB1 polypeptides and subjects (e.g. a human HMGB1 polypeptide for use in a chicken vaccine) may be useful in the methods of the invention because HMGB1 is highly conserved through a wide number of species. The HMGB1 polypeptide may be used to enhance the immune response in the subject to any foreign antigen, antigenic polypeptide or more than one polypeptide present in or on the vaccine vector. One of skill in the art will appreciate that the HMGB1 polypeptide could be used to enhance the immune response to more than one antigenic polypeptide present in a vaccine vector. The polypeptide from HMGB1 stimulates an immune response at least in part by activating dendritic cells and macrophages and thus stimulating production of cytokines such as IL-1, IL-6, IFN-γ and TNF-α. In the Examples, a polypeptide of HMGB1 was expressed on the surface of the vaccine vector.

The vaccine vector may suitably contain a CD154 polypeptide capable of binding to CD40 and activating CD40. The vaccine comprising the polynucleotide encoding a CD154 polypeptide capable of binding to CD40 is administered to a subject in an amount effective to enhance or effect the immune response of the subject to the vaccine. Suitably, the vaccine contains a polynucleotide encoding a polypeptide including amino acids 140-149 of the human CD154 polypeptide (SEQ ID NO: 25) or a homolog thereof. As noted above, a homologue of amino acid 140-149 derived from one species may be used to stimulate an immune response in a distinct species. Suitably, the polynucleotide encodes a CD154 polypeptide from the same species as the subject. Suitably, a polynucleotide encoding the polypeptide of SEQ ID NO: 26 is used in human subjects, a polynucleotide encoding the polypeptide of SEQ ID NO: 27 is used in chickens, a polynucleotide encoding the polypeptide of SEQ ID NO: 28 is used in ducks, a polynucleotide encoding the polypeptide of SEQ ID NO: 29 is used in mice, and a polynucleotide encoding the polypeptide of SEQ ID NO: 30 is used in cows. The human CD154 polypeptide (SEQ ID NO: 26) has been used in a chicken vaccine and was demonstrated to enhance the immune response to a foreign antigen. Thus other heterologous combinations of CD154 polypeptides and subjects may be useful in the methods of the invention.

In addition, methods of enhancing an immune response against a gram negative bacterium selected from Salmonella spp, Escherichia spp, Shigella spp, Vibrio spp, Erwinia spp, Klebsiella spp, Citrobacter spp, Yersinia spp, Providencia spp and similar bacteria and methods of reducing morbidity associated with subsequent infection with a gram-negative bacterium are disclosed. Briefly, the methods comprise administering to a subject a vaccine vector comprising a first polynucleotide sequence encoding an antigenic PAL polypeptide and optionally a second polynucleotide encoding an immunostimulatory polypeptide in an effective amount. The antigenic PAL polypeptides may include SEQ ID NO: 1-6. The insertion of the antigenic PAL polypeptides into the vector may be accomplished in a variety of ways known to those of skill in the art, including but not limited to the scarless site-directed mutation system described in BMC Biotechnol. 2007 Sep. 17: 7(1): 59, Scarless and Site-directed Mutagenesis in Salmonella Enteritidis chromosome, which is incorporated herein by reference in its entirety and the method used herein as described in Nguyen and Schumann J Biotechnol 2006 122: 473-482, which is incorporated herein by reference in its entirety. The vector may also be engineered to express the antigenic PAL polypeptides in conjunction with other antigenic polypeptides from other pathogens including viruses such as Influenza M2e or bacteria such as Salmonella, Campylobacter or E. coli. In particular, a polypeptide of CD154 capable of binding CD40 or HMGB1 may be expressed by the vector to enhance the immune response of the subject to the antigenic PAL polypeptide.

The compositions containing antigenic polypeptides may also be used to decrease the morbidity associated with subsequent infection by a gram-negative bacterium. The compositions may prevent the bacterium from causing disease or may limit or reduce any associated morbidity in a subject to which the compositions or vaccine vectors described herein were administered. The compositions and vaccine vectors described herein may reduce the severity of subsequent disease by decreasing the length of disease, weight loss, severity of symptoms of the disease, decreasing the morbidity or mortality associated with the disease or reducing the likelihood of contracting the disease. The compositions may also reduce the spread of the pathogen by inhibiting transmission. The morbidity or mortality associated with the disease after administration of the vaccine vectors described herein may be reduced by 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100% as compared to similar subjects not provided the vaccine vector.

For administration to animals or humans, the compositions may be administered by a variety of means including, but not limited to, intranasally, mucosally, by spraying, intradermally, parenterally, subcutaneously, intraperitonelly, intravenously, intracrannially, orally, by aerosol or intramuscularly. Eye-drop administration, oral gavage or addition to drinking water or food is additionally suitable. For poultry, the compositions may be administered in ovo.

Some embodiments of the invention provide methods of enhancing immune responses in a subject. Suitable subjects may include, but are not limited to, vertebrates, suitably mammals, suitably a human, and birds, suitably poultry such as chickens or turkeys. Other animals such as cows, cats, dogs or pigs may also be used. Suitably, the subject is non-human and may be an agricultural animal.

The useful dosage of the vaccine to be administered will vary depending on the age, weight and species of the subject, the mode and route of administration and the type of pathogen against which an immune response is sought. The composition may be administered in any dose sufficient to evoke an immune response. It is envisioned that doses ranging from 10³ to 10¹⁰ vector copies (i.e. colony forming units or plaque forming units), from 10⁴ to 10⁹ vector copies, or from 10⁵ to 10⁷ vector copies are suitable.

The composition may be administered only once or may be administered two or more times to increase the immune response. For example, the composition may be administered two or more times separated by one week, two weeks, three weeks, 1 month, 2 months, 3 months, 6 months, 1 year or more. The vaccine vector may comprise viable microorganisms prior to administration, but in some embodiments the vector may be killed prior to administration. In some embodiments, the vector may be able to replicate in the subject, while in other embodiments the vector may not be capable of replicating in the subject, e.g. a killed vaccine vector or a liposome. Methods of inactivating microorganisms used as vectors are known to those of skill in the art. For example, a bacterial vaccine vector may be inactivated using formalin, ethanol, heat exposure, or antibiotics. Those of skill in the art may use other methods as well.

It is envisioned that several epitopes or antigens from the same or different pathogens may be administered in combination in a single vaccine to generate an enhanced immune response against multiple antigens. Recombinant vaccines may encode antigens from multiple pathogenic microorganisms, viruses or tumor associated antigens. Administration of vaccine capable of expressing multiple antigens has the advantage of inducing immunity against two or more diseases at the same time. For example, live attenuated bacteria provide a suitable vector for eliciting an immune response against multiple antigens from a single pathogen, e.g., FliC and PAL from Salmonella or against multiple antigens from different pathogens, e.g., Influenza and Salmonella.

Vaccine vectors may be constructed using exogenous polynucleotides encoding antigens which may be inserted into the vaccine vector at any non-essential site or alternatively may be carried on a plasmid or other extra chromosomal vehicle (e.g. a BAC or YAC) using methods well known in the art. One suitable site for insertion of polynucleotides is within external portions of transmembrane proteins or coupled to sequences that target the exogenous polynucleotide for secretory pathways and/or allow attachment to the cell wall. One example of a suitable transmembrane protein for insertion of polynucleotides is the lamB gene. One suitable method of cell wall attachment is provided in the Examples

Exogenous polynucleotides include, but are not limited to, polynucleotides encoding antigens selected from pathogenic microorganisms or viruses and include polynucleotides that are expressed in such a way that an effective immune response is generated. Such polynucleotides may be derived from pathogenic viruses such as influenza (e.g., M2e, hemagglutinin, or neuraminidase), herpesviruses (e.g., the genes encoding the structural proteins of herpesviruses), retroviruses (e.g., the gp160 envelope protein), adenoviruses, paramyxoviruses, coronaviruses and the like. Exogenous polynucleotides can also be obtained from pathogenic bacteria, e.g., genes encoding bacterial proteins such as toxins, outer membrane proteins or other highly conserved proteins. Further, exogenous polynucleotides from parasites, such as Apicomplexan parasites are attractive candidates for use in a vector vaccine.

The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and/or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of” and “consisting of” those certain elements. The terms “a”, “an” and “the” may mean one or more than one unless specifically delineated.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims. All references, included patents, patent publications and non-patent literature, cited herein are hereby incorporated by reference in their entirety. Any conflict between statements in references and those made herein should be resolved in favor of the statements contained herein.

Examples

We selected the Pal polypeptide from E. coli as a highly conserved polypeptide that may include a polypeptide that would be both highly conserved among the gram-negative pathogenic bacteria and immunogenic. We began by selecting the E. coli sequence from amino acid 106-124 of Pal (P0A912). The antigenic potential of the selected sequence was confirmed using the Network Protein Sequence Analysis program against published sequences found in EMBL and NCBI databases (Combet, C., C. Blanchet, C. Geourjon, and G. Deleage. 2000. NPS@: network protein sequence analysis. Trends Biochem Sci 25:147-50). The sequence was then used to search for sequence homology using a Blast search engine on Swiss Institute of Bioinformatics on the EXPASY server. The Blast search found a number of proteins (Pal) with identical sequences to our initially selected Pal sequence (TVEGHADERGTPEYNISLG (SEQ ID NO: 8)). The list of Pal proteins with identical sequence include E. coli spp, Salmonella typhi and paratyphi spp, Shigella spp, Enterobacter spp, Citrobacter spp, Cronobacter spp. Also, Pal proteins with greater than 94% homology (only one amino acid different with or without similar substitution of a second amino acid) are Vibrio spp, Sodalis spp, Erwinia spp, Klebsiella spp, Dickeya spp, Serratia spp, Proteus spp, Xenorhabdus spp, Pectobacterium spp, and Pantoea spp with 100% coverage.

To optimize the antigen for other pathogen species, the 17^(th) amino acid will be changed from serine to alanine. The new sequence would be TVEGHADERGTPEYNIALG (SEQ ID NO: 32). This sequence is expected to provide optimal immune stimulation for Vibrio spp, Sodalis spp, Erwinia spp, Klebsiella spp, Dickeya spp, Serratia spp, Proteus spp, Xenorhabdus spp, Pectobacterium spp, and Pantoea spp with 100% coverage and either identical or similar amino acid sequence. The proteins of these species would be expected to be targeted by the immune system following vaccination and provide protection against these organisms.

The PAL epitope (TVEGHADERGTPEYNISLG (SEQ ID NO: 8)) was inserted into a Bacillus subtilis (BS) vector and expressed. The PAL Bacillus construct was then tested as a vaccine vector for Salmonella by vaccinating chicks via oral gavage with 10⁸ cfu/chick on the day of hatch and comparing to chicks treated similarly with the Bacillus backbone (BS BB) or saline. The birds were boosted with the same treatment at 11 days post-hatch. Samples were harvested for specific immune response on day 17. The immune response to the vaccine was evaluated by measuring serum IgG (FIG. 1) and secretory ileal IgA (FIG. 2). Following vaccination with the selected sequence of Pal expressed on the Bacillus there was a significant serum and secretory immune response specifically against the Pal sequence compared to controls (FIGS. 1 and 2).

Evaluation of potential Bacillus vectored vaccine candidates against Salmonella typhimurium (ST) challenge at 11 days post-vaccination was undertaken by enumerating Salmonella colonies in the ceca of vaccinated chickens at day 17 and 21 post-hatch (or day 6 and 10 after challenge). The levels of ST in the ceca were measured using conventional microbiological techniques. Chickens that were vaccinated with the selected sequence of Pal expressed on the Bacillus vector had significantly decreased levels of Salmonella in the ceca. As shown in FIG. 3, the level of Salmonella in the ceca was decreased by over 4½ logs in chicks vaccinated with BS-PAL (BSNNP) as compared to chicks vaccinate with saline or the BS BB. This is the first effective vaccine against Salmonella that is vectored by a Generally Recognized As Safe (GRAS) organism by the Food and Drug Administration (FDA) such as Bacillus subtilis.

In an investigation aimed at optimization of the immunogen sequence, referred to as PAL above (TVEGHADERGTPEYNISLG (SEQ ID NO: 8)), an epitope mapping experiment was designed to assess the relative antigenicity of portions of this 19-mer oligopeptide PAL. The sequence was split into 7 hexapeptides that overlapped by 3 amino acids each. For example, TVEGHA (SEQ ID NO: 39), GHADER (SEQ ID NO: 2), and DERGTP (SEQ ID NO: 3) each share three amino acids with the portion of sequence immediately to the left (toward the amino terminus) and right (toward the carboxy terminus). For this purpose, seven hexapeptides straddling amino acid residues 1-3, 4-6, 7-9 etc. were synthesized and coupled to bovine serum albumin (BSA). Two monoclonal antibodies (mAbs, designated 2B5 and 1B2) that reacted strongly with both the PAL 19-mer peptide and the native epitope as displayed on the cell wall of Salmonella (and related species) were selected and their relative affinities towards each segment of PAL were tested (FIG. 4).

The results indicated that, out of the 7 peptides tested, PAL1 (3 residues pre-PAL, “YKV”, and PAL amino terminal residues “TVE”; SEQ ID NO: 38) was the least antigenic for both mAbs. This can be explained by the observation that threonine is an uncharged amino acid and valine is an aliphatic residue, both of which are relatively hydrophobic and thus less likely to be accessible in the original immunogen PAL. Less accessible residues are unlikely to induce a potent immune response. In addition, most antibody epitopes are hydrophilic in nature. In contrast, the two best reacting mAbs had a much higher affinity for PAL6 (SEQ ID NO: 4) and PAL7 (SEQ ID NO: 6) (ELISA absorption levels compared to PAL1 were twice as high for PAL6 and >50% higher for PAL7). These results clearly indicate that the C-terminal half of PAL was likely the more exposed and accessible part of the immunogen and the most crucial portion of the immunogen with regard to generation of an antibody population that strongly cross-reacted with the native protein as displayed by Salmonella and related bacterial species. Interestingly, in order to generate the PAL7 hexapeptide, 2 residues were added that were not part of the original 19-mer PAL, but that flank PAL in the native bacterial protein: E (glutamate) and R (arginine). Both of these are charged residues and hence have a high probability of being exposed in our bacterial target species.

Based on the above rationale, a new 19-mer, designated PALbis (SEQ ID NO: 1), was generated. PALbis is different from the original PAL 19-mer in that (1) it no longer contains the two N-terminal amino acids T (threonine) and V (valine) and (2) it has been extended C-terminally with two additional residues, i.e. E (glutamate) and R (arginine). Thus, the improved amino acid sequence, PALbis, is EGHADERGTPEYNISLGER (SEQ ID NO: 1). PALbis was compared against multiple genera of bacteria to ensure cross-species reactivity was maintained (BLAST results are shown in Table 1). Sequence homology among E. coli, Salmonella typhi and paratyphi, Shigella, Enterobacter, Citrobacter, and Cronobacter spp. still had 100% homology. Sequence homology among Vibrio, Sodalis, Erwinia, Klebsiella, Dickeya, Serratia, Proteus, Xenorhabdus, Pectobacterium, and Pantoea spp. have 95% homology with a single amino acid substitution S15A (SEQ ID NO: 6). The related Campylobacter jejuni sequence is shown as SEQ ID NO: 7 and has 65% identity with the sequence of SEQ ID NO: 1. Thus we choose to pursue vaccine vectors expressing SEQ ID NO: 1 to obtain cross-strain immune responses with a single vaccine vector.

TABLE 1 Sequence comparison of PALbis (SEQ ID NO: 1) among bacteria PALbis Sequence: EGHADERGTPEYNI S LGER E. coli (SEQ ID NO: 1) EGHADERGTPEYNI A LGER Vibrio (SEQ ID NO: 6) EG NC DE W GT D EYN QA LG__ Campylobacter (SEQ ID NO: 7) Bacteria Homology (%) Escherichia colt 100 Salmonella enteriditis 100 Salmonella typhimurium 100 Salmonella choleraesuis 100 Salmonella enterica 100 subspecies Montevideo Salmonella enterica 100 subspecies Kentucky Shigella flexneri 100 Shigella dysenteriae 100 Enterobacter radicincitans 100 Enterobacter hormaechei 100 Enterobacter asburiae 100 Enterobacter cancerogenus 100 Enterobacter cloacae 100 Enterobacter aerogenes 95 Citrobacter koseri 100 Citrobacter freundii 100 Citrobacter rodentium 100 Citrobacter youngae 100 Vibrio cholera 95 Vibrio scophthalmi 95 Vibrio rotiferianus 95 Vibrio ichthyoenteri 95 Vibrio harceyi 95 Vibrio mimicus 95 Vibrio alginolyticus 95 Vibrio shilonii 95 Vibrio parahaemolyticus 95 Vibrio tubiashii 95 Vibrio sinaloensis 95 Vibrio brasiliesis 95 Vibrio caribbenthicus 95 Vibrio orientalis 95 Vibrio ordalii 95 Vibrio nigripulchritudo 95 Vibrio anguillarum 95 Vibrio furnissii 95 Vibrio metschnikovii 95 Vibrio coralliilyticus 95 Vibrio splendidus 95 Vibrio vulnificus 95 Cronobacter sakazakii 100 Sodalis glossinidius 95 Erwinia billingiae 95 Klebsiella oxytoca 95 Klebsiella pneumonia 95 Dickeya dadantii 95 Dickeya zeae 95 Serratia symbiotica 95 Serratia plymuthica 95 Serratia proteamaculans 95 Serratia odorifera 95 Proteus mirabilis 95 Proteus penneri 95 Xenorhabdus bovienii 95 Xenorhabdus nematophila 95 Pectobacterium wasabiae 95 Pectobacterium carotovorum 95 Pectobacerium atrosepticum 95 Pantoea stewartii 95 Pantoea ananatis 95 Campylobacter jejuni 65

To test the ability of PALbis (SEQ ID NO: 1) to work in a cross-strain challenge experiment, several vaccine candidates were generated. The vaccine vectors used herein were generated substantially as described in International Publication No. WO2008/036675 and International Publication No. WO2011/091255. Three separate constructs were generated and incorporated into two separate vaccine vectors, either Salmonella Enteriditis or Salmonella Typhimurium. The inserts used included a polynucleotide encoding the CJ0113 epitope described as SEQ ID NO: 31 herein and originally described in International Publication No. WO2011/156619, a polynucleotide encoding the HMGB1 polypeptide of SEQ ID NO: 24 which was originally described in International Publication No. WO2011/091255, and the PALbis sequence of SEQ ID NO: 1 identified and described herein. The three polynucleotides were separated by serine spacers (three serine residues inserted to avoid steric hindrance issues) and inserted in various orders in frame into external loop 9 of the Salmonella transmembrane protein lamB. The resulting nucleic acid and amino acid sequences of the inserts are shown in SEQ ID NO: 41-46. SEQ ID NO: 41 and 42 are the nucleic acid and amino acid sequences of the CJ0113-PAL-HMGB1 insert, respectively. SEQ ID NO: 43 and 44 are the nucleic acid and amino acid sequences of the CJ0113-HMGB1-PAL insert, respectively. SEQ ID NO: 45 and 46 are the nucleic acid and amino acid sequences of the HMGB1-CJ0113-PAL insert, respectively. The purpose of generating three vaccine vectors with the same inserts in a variety of orders was to control for any position or steric hindrance effects of the polypeptides interacting with unmapped surface moieties on the vector agents which could make the HMGB1 binding domain inaccessible to receptors on the host cells, or which might make surface-presented antigens inaccessible to the host immune cells.

Salmonella Enteriditis vectored vaccines reduced Salmonella Heidelberg recovery after challenge. Chicks were vaccinated with a Salmonella Enteriditis vectored vaccine that belongs to a heterologous Salmonella serogroup when compared to the Salmonella Heidelberg challenge strain to determine whether the PAL antigen would generate a cross Salmonella serogroup immune response. Live Salmonella Enteriditis-CJ0113-PAL-HMGB1, live Salmonella Enteriditis-CJ0113-HMGB1-PAL (which was later determined to contain two point mutations in HMGB1 and a frame-shift mutation in PAL resulting in the PAL epitope of SEQ ID NO: 35), and live Salmonella Enteriditis-HMGB1-CJ0113-PAL (with a later determined point mutation in HMGB1) vaccines were oral gavaged in 1-day-old chicks at 4×10⁸ cfu/chick. Chicks were challenged on day 7 with a Salmonella Heidelberg at 7×10⁶ cfu/chick by oral gavage. Salmonella Heidelberg colony forming units (cfu) per gram isolated from the ceca of 21-day-old broiler chick were determined. Salmonella Heidelberg cfu/g that were recovered from the ceca 14 days after challenge of live Salmonella Enteriditis-CJ0113-PAL-HMGB1 vaccinated chickens were significantly lower than from live Salmonella Enteriditis-CJ0113-HMGB1-PAL with two point mutations in HMGB1 and a frame-shift mutation in PAL vaccinated chickens, live Salmonella Enteriditis-HMGB1-CJ0113-PAL with a point mutation in HMGB1 vaccinated chickens, and non-vaccinated control chickens (FIG. 5; P=0.003).

Chicks were also vaccinated with glutaraldehyde-inactivated Salmonella Enteriditis vectored vaccines belonging to a heterologous Salmonella serogroup when compared to the Salmonella Heidelberg challenge to determine whether the PAL antigen would generate a cross Salmonella serogroup immune response. Glutaraldehyde-inactivated Salmonella Enteriditis-CJ0113-PAL-HMGB1, Salmonella Enteriditis-CJ0113-mHMGB1-mPAL (with point mutations in HMGB1 and a frameshift mutation in PAL), Salmonella Enteriditis-mHMGB1-CJ0113-PAL (with a point mutation in HMGB1) vaccines were adjuvated with mannosylated chitosan (as described in International Application No. PCT/US13/67212). The prepared vaccines were used to oral gavage 1-day-old chicks at 1×10⁹ cfu/chick. Chicks were challenged on day 17 with a Salmonella Heidelberg at 8.5×10⁶ cfu/chick by oral gavage. Glutaraldehyde-inactivated Salmonella Enteriditis-CJ0113-PAL-HMGB1 vaccination and Salmonella Enteriditis-mHMGB1-CJ0113-PAL vaccination in broilers significantly reduced Salmonella Heidelberg recovery from the ceca five days after challenge (FIG. 6; P<0.05), and Salmonella Heidelberg recovery remained low in Salmonella Enteriditis-mHMGB1-CJ0113-PAL and Salmonella Enteriditis-CJ0113-PAL-HMGB1 vaccinated chickens seventeen days after challenge (P=0.033). These data indicate that the PAL epitope in these vaccines provided protection against a cross-serogroup Salmonella challenge considering that the vaccine backbone originated from a Salmonella serogroup D strain and protected against a Salmonella serogroup B challenge.

Notably, these experiments were not useful to determine if there was any effect of the relative orientation or position of the three polypeptides in the vaccine vector because there were mutations discovered in the inserts. The mutations were informative regarding the protective or immunogenic portion of the PAL polypeptide. A single nucleotide deletion was found in the PAL polynucleotide of the Salmonella Enteriditis-CJ0113-mHMGB1-mPAL vaccine. The wild-type PAL nucleotide sequence is 5′-GAAGGTCACGCGGACGAACGTGGTACCCCGGAATACAACATCTCTCTGGGTGAA CGT-3′ (SEQ ID NO: 33; the guanine deleted in the mutant sequence is underlined) and the mutant PAL sequence found in the Salmonella Enteriditis-CJ0113-mHMGB1-mPAL is 5′-GAAGGTCACGCGGACGAACGTGGTACCCCGAATACAACATCTCTCTGGGTGAAC GT-3′ (SEQ ID NO: 34). The guanine deletion (underlined in the wild-type sequence) 31 base pairs into the PAL nucleotide sequence caused a frame-shift mutation that changed the last eight amino acids of the PAL peptide sequence. The wild-type PAL of SEQ ID NO: 1 becomes SEQ ID NO: 35 (EGHADERGTPNTTSLWVN; the last eight amino acids are underlined and are different than those found in SEQ ID NO: 1). The lack of development of an effective immune response by this mutant PAL is likely due to the loss of the last nine amino acids of PAL, which were shown to be important for development of an antibody response in FIG. 4 above. Thus a minimal PAL epitope may be SEQ ID NO: 36 (EYNISLGER) or its Vibrio counterpart SEQ ID NO: 37 (EYNIALGER).

The vaccines were remade to correct the mutations noted above. Once the mutations were corrected, live Salmonella Enteriditis-CJ0113-PAL-HMGB1, live Salmonella Enteriditis-HMGB1-CJ0113-PAL, and live Salmonella Typhimurium-HMGB1-CJ0113-PAL vaccination in broilers significantly reduced Salmonella Heidelberg recovery after enrichment with tetrathionate for 24 hours from broilers' ceca collected 10 days after challenge (FIG. 7; P<0.05). Day of hatch chicks were vaccinated with 10⁷ cfu of live-Salmonella Enteriditis-CJ0113-PAL-HMGB1, Salmonella Enteriditis-CJ0113-HMGB1-PAL, Salmonella Enteriditis-HMGB1-CJ0113-PAL, Salmonella Typhimurium-CJ0113-PAL-HMGB1, Salmonella Typhimurium-CJ0113-HMGB1-PAL, or Salmonella Typhimurium-HMGB1-CJ0113-PAL by oral gavage. An additional group of day of hatch chicks was vaccinated with 10⁶ cfu Salmonella Enteriditis-CJ0113-PAL-HMGB1 by oral gavage. Salmonella Enteriditis-CJ0113-PAL-HMGB1, Salmonella Enteriditis-CJ0113-HMGB1-PAL, Salmonella Enteriditis-HMGB1-CJ0113-PAL, Salmonella Typhimurium-CJ0113-PAL-HMGB1, Salmonella Typhimurium-CJ0113-HMGB1-PAL, or Salmonella Typhimurium-HMGB1-CJ0113-PAL vaccinated chickens were boosted at 14-days-old with 10⁷ cfu of the respective vaccine. Salmonella Enteriditis-CJ0113-PAL-HMGB1 vaccinated chickens that received 10⁶ cfu on day of hatch were boosted with 10⁸ cfu of Salmonella Enteriditis-CJ0113-PAL-HMGB1. Chickens were challenged on day 17 with 6×10⁶ cfu/chicken by oral gavage and the results are shown in FIG. 7 as percent challenge bacteria recovery. The results suggest that the position of each of the insert in the vaccine vector may affect the level of protection offered by the vaccine.

PAL expression on the Salmonella Enteriditis bacterial cell surface will directly interact with B lymphocytes to stimulate antibody production. HMGB1 expression on the Salmonella Enteriditis cell surface affects the percentage phagocytic uptake into murine macrophages (FIG. 8). Murine macrophages from the Raw 264 cell line were co-cultured with live Salmonella Enteriditis vaccine vector, Salmonella Enteriditis-CJ0113-PAL-HMGB1, Salmonella Enteriditis-CJ0113-HMGB1-PAL, or Salmonella Enteriditis-HMGB1-CJ0113-PAL for one hour. Escherichia coli pHrodo red bioparticles were added to each culture and incubated for two hours. After the bioparticles and bacteria are engulfed by the macrophage a phagosome is created. The phagosome fuses with a lysosome fuses acidifying the inside of the phagolysosome. The fluorescence intensity of the bioparticles increase as the pH becomes more acidic; therefore, the bioparticles within a phagolysosome will have higher fluorescence intensity. Salmonella Enteriditis-CJ0113-PAL-HMGB1 percentage phagocytic uptake was higher than Salmonella Enteriditis-HMGB1-CJ0113-PAL which was higher than Salmonella Enteriditis-CJ0113-HMGB1-PAL suggesting that HMGB1 at the end of the insert interacts favorably with the cell surface and enhances phagocytic uptake.

Based on these data, the linear display of chimeric DNA alters protein folding that is dependent upon the position of charged amino acids. Different linear combinations of antigens and immune stimulatory molecules will affect the spatial arrangement of each antigen and immune stimulatory on the bacterial cell surface. The protein expression of these linear combinations may differ for each bacteria species because the channel proteins on the bacteria cell surface creating steric hindrance with surrounding channel proteins. Reduced vaccine efficacy could be the result of unfavorable PAL or HMGB1 protein expression due to steric hindrance. 

We claim:
 1. A vaccine vector comprising a first polynucleotide encoding a first antigenic polypeptide consisting of PAL polypeptide SEQ ID NO: 1, an amino acid sequence having 90% or more homology to SEQ ID NO: 1 or an immunogenic fragment thereof at least six amino acids long, wherein the PAL polypeptide is expressed on the surface of the vaccine vector.
 2. The vaccine vector of claim 1, further comprising a second polynucleotide sequence encoding an immunostimulatory polypeptide selected from the group consisting of SEQ ID NOs: 15-30 and combinations thereof, wherein the immunostimulatory polypeptide is expressed on the surface of the vaccine vector.
 3. The vaccine vector of claim 2, wherein the vector comprises more than one copy of the first polynucleotide and/or more than one copy of the second polynucleotide sequence.
 4. The vaccine vector of claim 2, wherein the first polynucleotide sequence is linked in frame to the second polynucleotide sequence.
 5. The vaccine vector of claim 4, wherein the first polynucleotide and the second polynucleotide are linked via a spacer nucleotide.
 6. The vaccine vector of claim 1, wherein the vector is selected from the group consisting of a virus, a bacterium, a yeast, and a liposome.
 7. The vaccine vector of claim 6, wherein the vaccine vector is selected from the group consisting of Bacillus spp., Salmonella spp., Lactobacillus spp., and Escherichia spp.
 8. The vaccine vector of claim 2, further comprising a third polynucleotide encoding a second antigenic polypeptide.
 9. The vaccine vector of claim 8, wherein the second antigenic polypeptide is a polypeptide selected from SEQ ID NO: 7 or SEQ ID NO:
 31. 10. A pharmaceutical composition comprising the vaccine vector of claim 1 and a pharmaceutically acceptable carrier.
 11. A method of enhancing the immune response against a gram-negative bacterium in a subject comprising administering to the subject the vaccine vector of claim 1 in an amount effective to enhance the immune response of the subject to the gram-negative bacterium.
 12. The method of claim 11, wherein the enhanced immune response comprises an enhanced antibody response, an enhanced T cell response or both.
 13. A method of reducing morbidity associated with infection with a gram-negative bacterium in a subject comprising administering to the subject the vaccine vector of claim 1 in an amount effective to reduce the morbidity associated with subsequent infection of the subject with a gram-negative bacterium as compared to a control subject not administered the vaccine vector.
 14. The method of claim 11, wherein the vaccine vector is administered by a route selected from the group consisting of oral, mucosal, parenteral, sub-cutaneous, intramuscular, intraocular and in ovo.
 15. The method of claim 11, wherein the subject is selected from the group consisting of a poultry species and a mammal.
 16. The method of claim 15, wherein the subject is selected from the group consisting of a human, a chicken and a turkey.
 17. The method of claim 11, wherein from about 10⁴ to about 10⁹ vector copies of the vaccine are administered to the subject.
 18. The method claim 11, wherein the vaccine vector is killed prior to administration to the subject or is not capable of replicating in the subject.
 19. The method of claim 11, wherein the gram-negative bacterium is selected from the group consisting of Salmonella spp, Escherichia spp, Shigella spp, Vibrio spp, Erwinia spp, Klebsiella spp, Citrobacter spp, Yersinia spp, and Providencia spp.
 20. The method of claim 11, further comprising a second polynucleotide sequence encoding an immunostimulatory polypeptide selected from the group consisting of SEQ ID NOs: 15-30 and combinations thereof, wherein the immunostimulatory polypeptide is expressed on the surface of the vaccine vector. 